The monooxygenase system, which is composed of cytochrome P-450 and reductase, plays a key role in the metabolic activation of carcinogens. In this project, several approaches will be used to obtain decisive information concerning the structural organization of the monooxygenase and the subcellular site of carcinogen activation: 1. A chemical cross-linking approach will be be used to study the structural organization of monoxygenase enzymes in the membrane. 2. A "fluorescence recovery following photobleaching" method will be used to measure the lateral mobility of cytochrome P-450 in the membrane. 3. Temperature-jump and equilibrium binding studies will be used to study the effects of the phospholipids on the conformational state and substrate binding of P-450. 4. The covalent binding of benzo (a) pyrene and other carcinogens to nuclear macromolecules will be studied in reconstituted model systems to assess further the roles of nuclei, microsomes, and cytosol in the metabolic activation of carcinogens. 5. The kinetics and the extent of the covalent binding of carcinogens to microsomes and nuclei of isolated hepatocytes will be measured at different levels of carcinogen exposure to provide detailed information concerning the subcellular site of carcinogen activations.